Nucleic acid sequences encoding beta-ketoacyl-ACP synthase and uses thereof

ABSTRACT

This invention relates to sequences encoding β-ketoacyl-ACP synthase (KAS) and methods of use thereof. Also provided are methods for decreasing saturated fatty acid levels as a component of total triglycerides found in plant oils. The method generally comprises growing a soybean plant having integrated into its genome a DNA construct comprising, in the 5′ to 3′ direction of transcription, a promoter functional in a soybean plant seed cell, a DNA sequence encoding a KAS protein, and a transcription termination region functional in a plant cell. The present invention also provides a soybean seed with less than about 3.5 weight percent total saturated fatty acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. ProvisionalApplication Ser No. 60/220,702, filed Jul. 25, 2000, herein incorporatedby reference in its entirety.

TECHNICAL FIELD

[0002] The present invention is directed in general to β-ketoacycl-ACPsynthase nucleic acid and amino acid sequences and constructs, andmethods related thereto.

BACKGROUND OF THE INVENTION

[0003] Plant lipids have become indispensable in a number of industrialand nutritional applications. More importantly, plant lipids are usedextensively for their nutritional value, which is determined by aplant's fatty acid composition. The value of any plant lipid isdetermined by its chemical structure, which is a result of a plant'smetabolic processes. The chemical structure is characterized by varieddegrees of unsaturation. Most vegetable oils from commercial plantvarieties are composed primarily of palmitic (16:0), stearic (18:0),oleic (18:1), linoleic (18:2) and linolenic acid (18:3) acids.

[0004] Numerous research efforts have shown lipids to play a major rolein development of many diseases, especially cardiovascular disorders.Recent research has examined in great detail the role of saturated andunsaturated fatty acids in potentiating the risk of coronary heartdisease. Previously, it was believed that mono-unsaturated fatty acidshad no effect on serum cholesterol and coronary heart disease risk. Onthe other hand, saturated fatty acids were considered to contribute tocoronary heart disease while poly-unsaturated fatty acids were supposedto lower the risk of the same. It is now known that intake of bothmono-unsaturated and poly-unsaturated fatty acids is beneficial for theheart and overall health. Several recent human clinical studies suggestthat diets high in mono-unsaturated and/or poly-unsaturated fat and lowin saturated fat may reduce the “bad” (low-density lipoprotein or LDL)cholesterol while maintaining the “good” (high-density lipoprotein orHDL) cholesterol. For example, a study performed by Mensink et al.concluded that a diet rich in mono-unsaturated fatty acids was aseffective as a diet rich in (n-6)poly-unsaturated fats in lowering “bad”cholesterol (Mensink et al., N Engl J Med 321(7)436-441, August 1989).Furthermore, another study found that a diet rich in poly-unsaturatedfats has a similar effect on “good” cholesterol concentrations in theblood as a diet rich in mono-unsaturated fats (Dreon et al., JAMA263(18):2462-2466, May 9, 1990). Animal studies have also shown thatwhen monkeys are fed mono-unsaturated and poly-unsaturated fat diets,they have similar concentrations of LDL cholesterol, and these valuesare significantly lower than the LDL values from animals that are fedsaturated fats (Rudel et al., Arteriosclerosis, Thrombosis, and VascularBiology, 15:2101-2110, 1995).

[0005] Therefore, a vegetable oil low in total saturates and high inmono-unsaturates and/or poly-unsaturates would provide significanthealth benefits to all consumers. The beneficial effects of oils high inpoly-unsaturated fatty acids extend beyond lowering LDL cholesterol. Forinstance, linoleate and linolenate are essential fatty acids in humandiets, rendering any edible oil high in these fats a useful nutritionalsupplement. Certain plants naturally possess high levels ofpoly-unsaturated fatty acids. This is exemplified by linseed oil, whichis derived from the Flax plant (Linum usitatissimum) and contains over50% linolenic acid. The oil content of flax is comparable to canola(around 40% dry weight of seed), however, high yields are only obtainedin warm temperatures or subtropical climates. In addition, flax ishighly susceptible to rust infection in the U.S. Therefore, even thoughnatural plant sources of high poly-unsaturates exist, they are notalways useful for large scale oil production. It would be commerciallyuseful if a common crop such as canola, soybean or corn could begenetically transformed in such a way to minimize saturated fatty acidcontent.

[0006] To this effect, mutation-breeding programs have shown somepromise in altering the levels of poly-unsaturated fatty acid levels inthe edible oils of agronomic species. Examples of commercially grownvarieties are high (85%) oleic sunflower and low (2%) linolenic flax(Knowles, (1980) pp. 35-38 in Applewhite, T. E., Ed., World Conferenceon Biotechnology for the Fats and Oils Industry Proceedings, AmericanOil Chemists' Society). However, these breeding programs are difficultto maintain and yields are often low. Hence, the option of production oftransgenic plants is a desirable alternative for altering the content ofsaturated fats.

[0007] The enzymes of the fatty acid biosynthetic pathways are useful increating transgenic plants that have altered fatty acid content. Theβ-ketoacyl-ACP (acyl carrier protein) family of synthase enzymes (alsoreferred to herein as KAS) is especially attractive for planttransformation due to their indispensable role in fatty acid synthesis.To summarize their functions briefly, KAS III catalyzes the condensationof acetyl-CoA and malonyl-ACP to yield acetoacetyl-ACP in the firstelongating reaction, KAS I utilizes saturated C₂-C₁₄ and unsaturatedC_(16:1)-C_(18:1) acyl-ACPs as substrates for condensation with a C₂unit derived from malonyl-ACP; KAS II carries out the final extensionstep of unsaturated fatty acid biosynthesis (C_(16:1) to C_(18:1)) byutilizing C_(14:0) and C_(12:1)-C_(16:1) acyl-ACPs. KAS IV has asubstrate specificity between those of KAS III and KAS I, and is amedium chain specific condensing enzyme. See Siggaard-Andersen et al.,Proc. Natl. Acad. Sci., Vol. 91, pp. 11027-11031, November 1994, andDehesh et al., Plant J, 15(3):383-390, August 1998.

[0008] To elaborate, the biosynthesis of fatty acids is a complexprocess, involving numerous enzymes and multiple plant compartments. Theproduction of fatty acids in plants begins in the plastid with thereaction between acetyl-CoA and malonyl-ACP to produce butyryl-ACP.Elongation of acetyl-ACP to 16- and 18-carbon fatty acids involves thecyclical action of the following sequence of reactions: condensationwith a two-carbon unit from malonyl-ACP, reduction of the keto-functionto an alcohol, dehydration to form an enoyl-ACP, and finally reductionof the enoyl-ACP to form the elongated saturated acyl-ACP. KAS I,catalyzes elongation up to palmitoyl-ACP (C_(16:0)), whereas KAS IIcatalyzes the final elongation to stearoyl-ACP (C_(18:0)). The longestchain fatty acids produced by the fatty acid synthases are typically 18carbons long. A further fatty acid biochemical step occurring in theplastid is the desaturation of stearoyl-ACP (C_(8:0)) to form oleoyl-ACP(C_(18:1)) in a reaction catalyzed by a delta-9 desaturase.

[0009] Once the C_(18:1)-ACP has been formed, the products undergode-esterification, which allows movement into the cytoplasm, whereinthey are incorporated into the “eukaryotic” lipid biosynthesis pathway.This occurs in the endoplasmic reticulum, which is responsible for theformation of phospholipids, triglycerides and other neutral lipids.Following transport of fatty acyl CoA's to the endoplasmic reticulum,subsequent sequential steps for triglyceride production can take place.For example, polyunsaturated fatty acyl groups such as linoleoyl anda-linolenoyl, are produced as the result of sequential desaturation ofoleoyl acyl groups by the action of membrane-bound enzymes.Triglycerides are formed by action of the 1-, 2-, and 3-acyl-ACPtransferase enzymes glycerol-3-phosphate acyltransferase,lysophosphatidic acid acyltransferase and diacylglycerolacyltransferase. Alternatively, fatty acids are linked toglycerol-3-phosphate (prokaryotic path), further unsaturated, and usedfor synthesis of chloroplast lipids. A portion of cytoplasmic lipidsreturns to the chloroplast. The preferential use of either eukaryotic orprokaryotic pathway depends on the particular plant species. The fattyacid composition of a plant cell is a reflection of the free fatty acidpool and the fatty acids (fatty acyl groups) incorporated intotriglycerides as a result of the acyltransferase activities.

[0010] The properties of a given triglyceride will depend upon thevarious combinations of fatty acyl groups in the different positions inthe triglyceride molecule. In general, vegetable oils tend to bemixtures of different triglycerides. The triglyceride oil properties aretherefore a result of the combination of triglycerides which make up theoil, which are in turn influenced by their respective fatty acylcompositions.

[0011] There have been attempts to generate transgenic plants that wouldexhibit altered fatty acid levels for purposes of providing betternutritional value and herbicide and/or cold resistance. For instance,transformation of plants with maize acetyl CoA carboxylase or delta-15desaturase can alter the fatty acid content in the plants. See U.S. Pat.Nos. 6,222,099 and 5,952,544 respectively. Transformation of plants withmaize acetyl CoA carboxylase gene allows for altering the total oilcontent in the plant. However, this process does not provide a methodfor specifically increasing only the levels of unsaturated fats, whichwould in turn decrease the levels of saturated fats in the transgenicplants. U.S. Pat. No. 5,500,361 describes a nucleotide sequence thatencodes for a β-ketoacyl-ACP synthase II enzyme. However, the disclosedcompositions are limited in their use. Similarly, U.S. Pat. No.6,200,788, also discloses a nucleotide sequence that encodes for aspecific β-ketoacyl-ACP synthase II enzyme and has the same limitations.

[0012] Depending upon the intended oil use, various different oilcompositions are desired. For example, edible oil sources containing theminimum possible amounts of saturated fatty acids are desired fordietary reasons and alternatives to current sources of highly saturatedoil products, such as tropical oils, are also needed. Furthermore, oilscompositions containing rare or exotic fatty acid species havingnutritional benefits are also needed in the art. To this end, therefore,novel vegetable oils compositions and/or improved means to obtain ormanipulate fatty acid compositions, from biosynthetic or natural plantsources, are needed.

[0013] Accordingly, a need still exists to produce transgenic plantsthat would possess lower levels of saturated fats compared to the levelsin untransformed plants. These transgenic plants would provide avaluable nutritional supplement, especially if the oils extracted fromthem were incorporated into diets of people who were at risk for orsuffered from cardiovascular diseases. Thus a need exists for amulti-functional KAS synthase that can effectively alter the saturatedfatty acid content of a commercial plant. Thus, the identification ofnucleic acid sequences encoding enzymes capable of producing alteredsaturated fatty acid compositions in host cells is needed in the art.Ultimately, useful nucleic acid constructs having the necessary elementsto provide a phenotypic modification and host cells containing suchconstructs are needed.

SUMMARY OF THE INVENTION

[0014] The present invention is directed to fatty acid synthase, and inparticular to β-ketoacyl-ACP synthase (also referred to herein as KAS)polypeptides and polynucleotides. In one aspect of the presentinvention, the polypeptides and polynucleotides are derived fromcyanobacterial sources.

[0015] In another aspect of the invention polynucleotides encoding novelpolypeptides, particularly, polynucleotides that encode β-ketoacyl-ACPsynthase, are provided.

[0016] In a further aspect the invention relates to oligonucleotidesderived from the β-ketoacyl-ACP synthase proteins and oligonucleotideswhich include partial or complete β-ketoacyl-ACP synthase encodingsequences.

[0017] It is also an aspect of the present invention to providerecombinant DNA constructs which can be used for transcription ortranscription and translation (expression) of β-ketoacyl-ACP synthase.In particular, constructs are provided which are capable oftranscription or transcription and translation in host cells.Particularly preferred constructs are those capable of transcription ortranscription and translation in host plant cells.

[0018] In another aspect of the present invention, methods are providedfor production of β-ketoacyl-ACP synthase in a host cell or progenythereof. In particular, host cells are transformed or transfected with aDNA construct which can be used for transcription or transcription andtranslation of β-ketoacyl-ACP synthase. The recombinant cells whichcontain β-ketoacyl-ACP synthase are also part of the present invention.

[0019] In a further aspect, the present invention relates to methods ofusing polynucleotide and polypeptide sequences to modify the fatty acidcomposition in a host cell, particularly the fatty acid composition inthe seed tissue of host plants. In particular, host plants such asBrassica, corn and soybean. And in further aspect of the invention, themodified fatty acid composition comprises an altered amount of saturatedfatty acids. Plant cells having such a modified fatty acids are alsocontemplated herein.

[0020] The modified plants, seeds, their progeny and oils obtained bythe expression of the plant β-ketoacyl-ACP synthase proteins are alsoconsidered part of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 provides a graphic representation of the reduction of totalsaturated fatty acids (C6:0 to C24:0) in pooled seed of Brassica plantscontaining the Cpu KASI/CpuKASIV and safflower delta-9 desaturase.

[0022]FIG. 2 provides a table of fatty acid analysis of oil from soybeantransformed with the construct pCGN9807.

[0023]FIG. 3 provides a graphic representation of the C16:0 fatty acidlevels in soybean oil from plants containing the expression constructpCGN9807.

[0024]FIG. 4 provides a graphic representation of the C20:1 fatty acidlevels in soybean oil from plants containing the expression constructpCGN9807.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The following detailed description is provided to aid thoseskilled in the art in practicing the present invention. Even so, thisdetailed description should not be construed to unduly limit the presentinvention as modifications and variations in the embodiments discussedherein can be made by those of ordinary skill in the art withoutdeparting from the spirit or scope of the present inventive discovery.

[0026] All publications, databases, patents, patent applications andother references cited in this application are herein incorporated byreference in their entirety as if each individual publication, patent,patent application or other reference were specifically and individuallyindicated to be incorporated by reference.

[0027] In accordance with the subject invention, isolated nucleotidesequences are provided which are capable of encoding sequences of aminoacids, such as, a protein, polypeptide or peptide, which encodeβ-ketoacyl-ACP synthase (also referred to herein as KAS). The novelnucleic acid sequences find use in the preparation of constructs todirect their expression in a host cell. Furthermore, the novel nucleicacid sequences find use in the preparation of plant expressionconstructs to modify the saturated fatty acid composition of a hostcell. Also provided are plant oil compositions comprising a low level ofsaturated fatty acids.

[0028] Isolated Proteins, Polypeptides and Polynucleotides

[0029] A first aspect of the present invention relates to isolatedβ-ketoacyl-ACP synthase polynucleotides. The polynucleotide sequences ofthe present invention include isolated polynucleotides that encode thepolypeptides of the invention having a deduced amino acid sequence setforth in SEQ ID NO: 2 and to other polynucleotide sequences closelyrelated to such sequences and variants thereof.

[0030] The invention provides a polynucleotide sequence identical overits entire length to each coding sequence as set forth in SEQ ID NO: 1.The invention also provides the coding sequence for the maturepolypeptide or a fragment thereof, as well as the coding sequence forthe mature polypeptide or a fragment thereof in a reading frame withother coding sequences, such as those encoding a leader or secretorysequence, a pre-, pro-, or pre-pro-protein sequence. The polynucleotidecan also include non-coding sequences, including for example, but notlimited to, non-coding 5′ and 3′ sequences, such as the transcribed,untranslated sequences, termination signals, ribosome binding sites,sequences that stabilize mRNA, introns, polyadenylation signals, andadditional coding sequences that encode additional amino acids. Forexample, a marker sequence can be included to facilitate thepurification of the fused polypeptide. Polynucleotides of the presentinvention also include polynucleotides comprising a structural gene andthe naturally associated sequences that control gene expression.

[0031] The invention also includes polynucleotides of the formula:

X—(R₁)_(n)—(R₂)—(R₃)_(n)—Y

[0032] wherein, at the 5′ end, X is hydrogen, and at the 3′ end, Y ishydrogen or a metal, R₁ and R₃ are any nucleic acid residue, n is aninteger between 1 and 3000, preferably between 1 and 1000 and R₂ is anucleic acid sequence of the invention, particularly a nucleic acidsequence as set forth in SEQ ID NO: 1. In the formula, R₂ is oriented sothat its 5′ end residue is at the left, bound to R₁, and its 3′ endresidue is at the right, bound to R₃. Any stretch of nucleic acidresidues denoted by any R group, R₁, R₂ or R₃, and where R is greaterthan 1, may be either a heteropolymer or a homopolymer, preferably aheteropolymer.

[0033] The invention also relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptides of theinvention. Variants that are fragments of the polynucleotides of theinvention can be used to synthesize full-length polynucleotides of theinvention. Preferred embodiments are polynucleotides encodingpolypeptide variants wherein 5 to 10, 1 to 5, 1 to 3, 2, 1 or no aminoacid residues of a polypeptide sequence of the invention aresubstituted, added or deleted, in any combination. Particularlypreferred are substitutions, additions, and deletions that are silentsuch that they do not alter the properties or activities of thepolynucleotide or polypeptide.

[0034] Nucleotide sequences encoding β-ketoacyl-ACP synthase may beobtained from natural sources or be partially or wholly artificiallysynthesized. They may directly correspond to a KAS endogenous nucleotidesequence or contain modified amino acid sequences, such as sequenceswhich have been mutated, truncated, increased or the like.β-ketoacyl-ACP synthase may be obtained by a variety of methods,including but not limited to, partial or homogenous purification ofprotein extracts, protein modeling, nucleic acid probes, antibodypreparations and sequence comparisons. Typically a KAS will be derivedin whole or in part from a natural source. A natural source includes,but is not limited to, prokaryotic and eukaryotic sources, including,bacteria, yeasts, plants, including algae, and the like.

[0035] Of special interest are β-ketoacyl-ACP synthases which areobtainable from cyanobacterial sources, including those which areobtained, from Synechocystis, or from additional sources which areobtainable through the use of these sequences. “Obtainable” refers tothose KAS's which have sufficiently similar sequences to that of thesequences provided herein to provide a biologically active protein ofthe present invention.

[0036] Further preferred embodiments of the invention that are at least50%, 60%, or 70% identical over their entire length to a polynucleotideencoding a polypeptide of the invention, and polynucleotides that arecomplementary to such polynucleotides. More preferable arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding a polypeptide of theinvention and polynucleotides that are complementary thereto. In thisregard, polynucleotides at least 90% identical over their entire lengthare particularly preferred, those at least 95% identical are especiallypreferred. Further, those with at least 97% identity are highlypreferred and those with at least 98% and 99% identity are particularlyhighly preferred, with those at least 99% being the most highlypreferred.

[0037] Preferred embodiments are polynucleotides that encodepolypeptides that retain substantially the same biological function oractivity as the mature polypeptides encoded by the polynucleotide setforth in SEQ ID NO: 1.

[0038] The invention further relates to polynucleotides that hybridizeto the above-described sequences. In particular, the invention relatesto polynucleotides that hybridize under stringent conditions to theabove-described polynucleotides. As used herein, the terms “stringentconditions” and “stringent hybridization conditions” mean thathybridization will generally occur if there is at least 95% andpreferably at least 97% identity between the sequences. An example ofstringent hybridization conditions is overnight incubation at 42° C. ina solution comprising 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmonsperm DNA, followed by washing the hybridization support in 0.1×SSC atapproximately 65° C. Other hybridization and wash conditions are wellknown and are exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989),particularly Chapter 11.

[0039] The invention also provides a polynucleotide consistingessentially of a polynucleotide sequence obtainable by screening anappropriate library containing the complete gene for a polynucleotidesequence set forth in SEQ ID NO: 1 under stringent hybridizationconditions with a probe having the sequence of said polynucleotidesequence or a fragment thereof; and isolating said polynucleotidesequence. Fragments useful for obtaining such a polynucleotide include,for example, probes and primers as described herein.

[0040] As discussed herein regarding polynucleotide assays of theinvention, for example, polynucleotides of the invention can be used asa hybridization probe for RNA, cDNA, or genomic DNA to isolate fulllength cDNAs or genomic clones encoding a polypeptide and to isolateCDNA or genomic clones of other genes that have a high sequencesimilarity to the polynucleotide set forth in SEQ ID NO: 1. Such probeswill generally comprise at least about 15 bases. Preferably such probeswill have at least about 30 bases and can have at least about 50 bases.Particularly preferred probes will have between about 30 bases and about50 bases, inclusive.

[0041] The coding region of each gene that comprises or is comprised bythe polynucleotide sequence set forth in SEQ ID NO: 1 may be isolated byscreening using the DNA sequence provided in SEQ ID NO: 1 to synthesizean oligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to identify members of thelibrary which hybridize to the probe. For example, syntheticoligonucleotides are prepared which correspond tothe N-terminal sequenceof the polypeptide. The partial sequences so prepared can then be usedas probes to obtain KAS clones from a gene library prepared from a cellsource of interest. Alternatively, where oligonucleotides of lowdegeneracy can be prepared from particular peptides, such probes may beused directly to screen gene libraries for gene sequences. Inparticular, screening of cDNA libraries in phage vectors is useful insuch methods due to lower levels of background hybridization.

[0042] Typically, a sequence obtainable from the use of nucleic acidprobes will show about 60-70% sequence identity between the target KASsequence and the encoding sequence used as a probe. However, lengthysequences with as little as about 50-60% sequence identity may also beobtained. The nucleic acid probes may be a lengthy fragment of thenucleic acid sequence, or may also be a shorter, oligonucleotide probe.When longer nucleic acid fragments are employed as probes (greater thanabout 100 bp), one may screen at lower stringencies in order to obtainsequences from the target sample which have about 20-50% deviation(i.e., about a 50-80% sequence homology) from the sequences used asprobe. Oligonucleotide probes can be considerably shorter than theentire nucleic acid sequence encoding a KAS enzyme, but should be atleast about 10, preferably at least about 15, and more preferably atleast about 20 nucleotides. A higher degree of sequence identity isdesired when shorter regions are used as opposed to longer regions. Itmay thus be desirable to identify regions of highly conserved amino acidsequence to design oligonucleotide probes for detecting and recoveringother related genes. Shorter probes are often particularly useful forpolymerase chain reactions (PCR), especially when highly conservedsequences can be identified. (See, Gould, et al., PNAS USA (1989)86:1934-1938).

[0043] The skilled artisan will appreciate that, in many cases, anisolated cDNA sequence will be incomplete, in that the region coding forthe polypeptide is truncated with respect to the 5′ terminus of thecDNA. This is a consequence of the reverse transcriptase, an enzyme withlow ‘processivity’ (a measure of the ability of the enzyme to remainattached to the template during the polymerization reaction) employedduring the first strand cDNA synthesis.

[0044] There are several methods available and are well know to theskilled artisan to obtain full-length cDNAs, or extend short cDNAs; forexample those based on the method of Rapid Amplification of cDNA Ends(RACE) (see, for example, Frohman et al. (1988) Proc. Natl. Acad. Sci.USA 85:8998-9002). Recent modifications of the technique, exemplified bythe Marathonä technology (Clonetech Laboratories, Inc.) for example,have significantly simplified obtaining full-length cDNA sequences.

[0045] Another aspect of the present invention relates to isolatedβ-ketoacyl-ACP synthase polypeptides. Such polypeptides include theisolated polypeptide set forth in SEQ ID NO: 2, as well as polypeptidesand fragments thereof, particularly those polypeptides which exhibit KASactivity and also those polypeptides which have at least about 50%, 60%or 70% identity, preferably at least about 80% identity, more preferablyat least about 90% identity, and most preferably at least about 95%identity to the polypeptide sequence set forth in SEQ ID NO: 2, and alsoinclude portions of such polypeptides, wherein such portion of thepolypeptide preferably includes at least about 30 amino acids and morepreferably includes at least about 50 amino acids.

[0046] “Identity”, as is well understood in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as determined by the matchbetween strings of such sequences. “Identity” can be readily calculatedby known methods including, but not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York (1988); Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis ofSequence Data, Part I, Griffin, A. M. and Griffin, H. G., eds., HumanaPress, New Jersey (1994); Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov,M. and Devereux, J., eds., Stockton Press, New York (1991); and Carillo,H., and Lipman, D., SIAM J Applied Math, 48:1073 (1988). Methods todetermine identity are designed to give the largest match between thesequences tested. Moreover, methods to determine identity are codifiedin publicly available programs. Computer programs which can be used todetermine identity between two sequences include, but are not limitedto, GCG (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984);suite of five BLAST programs, three designed for nucleotide sequencesqueries (BLASTN, BLASTX, and TBLASTX) and two designed for proteinsequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology,12: 76-80 (1994); Birren, et al., Genome Analysis, 1: 543-559 (1997)).The BLAST X program is publicly available from NCBI and other sources(BLAST Manual, Altschul, S., et al., NCBI NLM NIH, Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol., 215:403-410 (1990)). The well knownSmith Waterman algorithm can also be used to determine identity.

[0047] Parameters for polypeptide sequence comparison typically includethe following:

[0048] Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)

[0049] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci USA 89:10915-10919 (1992)

[0050] Gap Penalty: 12

[0051] Gap Length Penalty: 4

[0052] A program which can be used with these parameters is publiclyavailable as the “gap” program from Genetics Computer Group, MadisonWis. The above parameters along with no penalty for end gap are thedefault parameters for peptide comparisons.

[0053] Parameters for polynucleotide sequence comparison include thefollowing:

[0054] Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)

[0055] Comparison matrix: matches=+10; mismatches=0

[0056] Gap Penalty: 50

[0057] Gap Length Penalty: 3

[0058] A program which can be used with these parameters is publiclyavailable as the “gap” program from Genetics Computer Group, MadisonWis. The above parameters are the default parameters for nucleic acidcomparisons.

[0059] The invention also includes polypeptides of the formula:

X—(R₁)_(n)—(R₂)—(R₃)_(n)—Y

[0060] wherein, at the amino terminus, X is hydrogen, and at thecarboxyl terminus, Y is hydrogen or a metal, R₁ and R₃ are any aminoacid residue, n is an integer between 1 and 1000, and R₂ is an aminoacid sequence of the invention, particularly the amino acid sequence ofSEQ ID NO: 2. In the formula, R₂ is oriented so that its amino terminalresidue is at the left, bound to R₁, and its carboxy terminal residue isat the right, bound to R₃. Any stretch of amino acid residues denoted byeither R group, where R is greater than 1, may be either a heteropolymeror a homopolymer, preferably a heteropolymer.

[0061] Polypeptides of the present invention include isolatedpolypeptides encoded by a polynucleotide comprising the polypeptidesequence of SEQ ID NO: 2.

[0062] Polypeptides of the present invention have been shown to haveβ-ketoacyl-ACP synthase activity and are of interest because KAS isinvolved in the elongation of acetyl-CoA through a condensation reactionwith a 2-carbon unit from malonyl-ACP to form β-ketoacyl-ACP. The KASproteins encoded by the nucleic acid sequences of the present inventiondemonstrate the ability to elongate various acyl chain lengths by twocarbon atoms. Thus the proteins of the present invention are useful incarrying out the condensation of saturated C₂-C₁₄ and unsaturatedC_(16:1)-C_(18:1) a C₂ unit derived from malonyl-ACP. Furthermore, theprotein of the instant invention is useful in carrying out the finalelongation of palmitoyl-ACP (C_(16:0)) to stearoyl-ACP (C_(18:0)).

[0063] Fatty acid biosynthesis in higher plants is catalyzed by a set ofenzymes located in plastids. Once malonyl-CoA is produced by acetyl-CoAcarboxylase (ACCase), the fatty acid synthase (FAS) transfers themalonyl moiety to ACP to use it as a carbon source for the synthesis oflong chain fatty acids, mainly 16:0 and 18:0. Each cycle of C₂ additionis initiated by a reaction catalyzed by a β-ketoacyl-ACP synthase (KAS)and involves the condensation of a malonyl-ACP with an acyl acceptor.The discovery and subsequent studies of KAS III resulted in significantchanges in the understanding of the initial reaction of the fatty acidbiosynthesis in plants. The in vitro (Jaworski et al., 1989; Clough etal., 1992) and in vivo (Jaworski et al., 1993) studies established thatKAS III initiates the fatty acid synthesis in plants, by catalyzing thecondensing reaction of acetyl-CoA and malonyl ACP. Subsequentcondensation reactions are catalyzed by other members of KAS family,namely KAS I, II and IV (Shimakata and Stumpf, 1982; Kauppinen et al.,1988; Dehesh et al., 1998)

[0064] The polypeptides of the present invention can be mature proteinsor can be part of a fusion protein. Fusion proteins can be useful inovercoming certain difficulties that are associated with expression offoreign proteins in the host cells. For example, if the desired proteinis degraded or produced in low quantities in the host, it can be fusedto a carrier (either a peptide or a protein) that is stable in the hostand produced in large quantities or simply produced in large quantities.In this way, an improved stability and higher expression of the desiredprotein can be achieved. The production of fusion proteins utilizesrecombinant DNA techniques previously described in the art and known toa skilled artisan.

[0065] Fragments and variants of the polypeptides are also considered tobe a part of the invention. A fragment is a variant polypeptide whichhas an amino acid sequence that is entirely the same as part but not allof the amino acid sequence of the previously described polypeptides. Thefragments can be “free-standing” or comprised within a largerpolypeptide of which the fragment forms a part or a region, mostpreferably as a single continuous region. Preferred fragments arebiologically active fragments which are those fragments that mediateactivities of the polypeptides of the invention, including those withsimilar activity or improved activity or with a decreased activity. Alsoincluded are those fragments that are antigenic or immunogenic in ananimal, particularly a human.

[0066] Variants of the polypeptide also include polypeptides that varyfrom the sequence set forth in SEQ ID NO: 2 by conservative amino acidsubstitutions, substitution of a residue by another with likecharacteristics. In general, such substitutions are among Ala, Val, Leuand Ile; between Ser and Thr; between Asp and Glu; between Asn and Gln;between Lys and Arg; or between Phe and Tyr. Particularly preferred arevariants in which 5 to 10; 1 to 5; 1 to 3 or one amino acid(s) aresubstituted, deleted, or added, in any combination.

[0067] Variants that are fragments of the polypeptides of the inventioncan be used to produce the corresponding full length polypeptide bypeptide synthesis. Therefore, these variants can be used asintermediates for producing the full-length polypeptides of theinvention.

[0068] The polynucleotides and polypeptides of the invention can beused, for example, in the transformation of various host cells, asfurther discussed herein.

[0069] The invention also provides polynucleotides that encode apolypeptide that is a mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids within the maturepolypeptide (for example, when the mature form of the protein has morethan one polypeptide chain). Such sequences can, for example, play arole in the processing of a protein from a precursor to a mature form,allow protein transport, shorten or lengthen protein half-life, orfacilitate manipulation of the protein in assays or production. It iscontemplated that cellular enzymes can be used to remove any additionalamino acids from the mature protein.

[0070] A precursor protein, having the mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. The inactive precursors generally are activated when theprosequences are removed. Some or all of the prosequences may be removedprior to activation. Such precursor protein are generally calledproproteins.

[0071] The polynucleotide and polypeptide sequences can also be used toidentify additional sequences which are homologous to the sequences ofthe present invention. The most preferable and convenient method is tostore the sequence in a computer readable medium, for example, floppydisk, CD ROM, hard disk drives, external disk drives and DVD, and thento use the stored sequence to search a sequence database with well knownsearching tools. Examples of public databases include the DNA Databaseof Japan (DDBJ)(http://www.ddbi.nig.ac.jp/); Genebank(http://www.ncbi.nlm.nih.gov/web/Genbank/Index.htlm); and the EuropeanMolecular Biology Laboratory Nucleic Acid Sequence Database (EMBL)(http://www.ebi.ac.uk/ebi docs/embl db.html). A number of differentsearch algorithms are available to the skilled artisan, one example ofwhich are the suite of programs referred to as BLAST programs. There arefive implementations of BLAST, three designed for nucleotide sequencesqueries (BLASTN, BLASTX, and TBLASTX) and two designed for proteinsequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology,12: 76-80 (1994); Birren, et al., Genome Analysis, 1: 543-559 (1997)).Additional programs are available in the art for the analysis ofidentified sequences, such as sequence alignment programs, programs forthe identification of more distantly related sequences, and the like,and are well known to the skilled artisan.

[0072] Plant Constructs and Methods of Use

[0073] Of interest in the present invention, is the use of thenucleotide sequences, or polynucleotides, in recombinant DNA constructsto direct the transcription or transcription and translation(expression) of the β-ketoacyl-ACP synthase sequences of the presentinvention in a host cell.

[0074] As used herein, “recombinant” includes reference to a cell orvector, that has been modified by the introduction of a heterologousnucleic acid sequence or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found in identical form within the native (non-recombinant) form ofthe cell or express native genes that are otherwise abnormallyexpressed, under expressed or not expressed at all as a result ofdeliberate human intervention.

[0075] Of particular interest is the use of the nucleotide sequences, orpolynucleotides, in recombinant DNA constructs to direct thetranscription or transcription and translation (expression) of the KASsequences of the present invention in a host plant cell. The expressionconstructs generally comprise a promoter functional in a host celloperably linked to a nucleic acid sequence encoding a KAS of the presentinvention and a transcriptional termination region functional in a hostcell.

[0076] By “host cell” is meant a cell which contains a vector andsupports the replication, and/or transcription or transcription andtranslation (expression) of the expression construct. Host cells for usein the present invention can be prokaryotic cells, such as E. coli, oreukaryotic cells such as yeast, plant, insect, amphibian, or mammaliancells. Preferably, host cells are monocotyledenous or dicotyledenousplant cells.

[0077] Those skilled in the art will recognize that there are a numberof promoters which are functional in plant cells, and have beendescribed in the literature. Chloroplast and plastid specific promoters,chloroplast or plastid functional promoters, and chloroplast or plastidoperable promoters are also envisioned.

[0078] One set of promoters are constitutive promoters such as theCaMV35S or FMV35S promoters that yield high levels of expression in mostplant organs. Enhanced or duplicated versions of the CaMV35S and FMV35Spromoters are useful in the practice of this invention (Odell, et al.(1985) Nature 313:810-812; Rogers, U.S. Pat. No. 5,378,619). Inaddition, it may also be preferred to bring about expression of theprotein of interest in specific tissues of the plant, such as leaf,stem, root, tuber, seed, fruit, etc., and the promoter chosen shouldhave the desired tissue and developmental specificity.

[0079] Of particular interest is the expression of the nucleic acidsequences of the present invention from transcription initiation regionswhich are preferentially expressed in a plant seed tissue. Examples ofsuch seed preferential transcription initiation sequences include thosesequences derived from sequences encoding plant storage protein genes orfrom genes involved in fatty acid biosynthesis in oilseeds. Examples ofsuch promoters include the 5′ regulatory regions from such genes asnapin (Kridl et al., Seed Sci. Res. 1:209:219 (1991)), phaseolin, zein,soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, soybean a′subunit of b-conglycinin (soy 7s, (Chen et al., Proc. Natl. Acad. Sci.,83:8560-8564 (1986))) and oleosin.

[0080] It may be advantageous to direct the localization of proteinsconferring KAS to a particular subcellular compartment, for example, tothe mitochondrion, endoplasmic reticulum, vacuoles, chloroplast or otherplastidic compartment. For example, where the genes of interest of thepresent invention will be targeted to plastids, such as chloroplasts,for expression, the constructs will also employ the use of sequences todirect the gene to the plastid. Such sequences are referred to herein aschloroplast transit peptides (CTP) or plastid transit peptides (PTP). Inthis manner, where the gene of interest is not directly inserted intothe plastid, the expression construct will additionally contain a geneencoding a transit peptide to direct the gene of interest to theplastid. The chloroplast transit peptides may be derived from the geneof interest, or may be derived from a heterologous sequence having aCTP. Such transit peptides are known in the art. See, for example, VonHeijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al.(1989) J. Biol. Chem. 264:17544-17550; della-Cioppa et al. (1987) PlantPhysiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res Commun.196:1414-1421; and, Shah et al. (1986) Science 233:478-481. Additionaltransit peptides for the translocation of the protein to the endoplasmicreticulum (ER) (Chrispeels, K., (1991) Ann. Rev. Plant Phys. Plant Mol.Biol. 42:21-53), nuclear localization signals (Raikhel, N. (1992) PlantPhys. 100:1627-1632), or vacuole may also find use in the constructs ofthe present invention.

[0081] Depending upon the intended use, the constructs may contain thenucleic acid sequence which encodes the entire KAS protein, or a portionthereof. For example, where antisense inhibition of a given KAS proteinis desired, the entire KAS sequence is not required. Furthermore, whereKAS sequences used in constructs are intended for use as probes, it maybe advantageous to prepare constructs containing only a particularportion of a KAS encoding sequence, for example a sequence which isdiscovered to encode a highly conserved KAS region.

[0082] The skilled artisan will recognize that there are various methodsfor the inhibition of expression of endogenous sequences in a host cell.Such methods include, but are not limited to antisense suppression(Smith, et al. (1988) Nature 334:724-726), co-suppression (Napoli, etal. (1989) Plant Cell 2:279-289), ribozymes (PCT Publication WO97/10328), and combinations of sense and antisense Waterhouse, et al.(1998) Proc. Natl. Acad. Sci. USA 95:13959-13964. Methods for thesuppression of endogenous sequences in a host cell typically employ thetranscription or transcription and translation of at least a portion ofthe sequence to be suppressed. Such sequences may be homologous tocoding as well as non-coding regions of the endogenous sequence.

[0083] Regulatory transcript termination regions may be provided inplant expression constructs of this invention as well. Transcripttermination regions may be provided by the DNA sequence encoding theβ-ketoacyl-ACP synthase or a convenient transcription termination regionderived from a different gene source, for example, the transcripttermination region which is naturally associated with the transcriptinitiation region. The skilled artisan will recognize that anyconvenient transcript termination region which is capable of terminatingtranscription in a plant cell may be employed in the constructs of thepresent invention.

[0084] Alternatively, constructs may be prepared to direct theexpression of the KAS sequences directly from the host plant cellplastid. Such constructs and methods are known in the art and aregenerally described, for example, in Svab, et al. (1990) Proc. Natl.Acad. Sci. USA 87:8526-8530 and Svab and Maliga (1993) Proc. Natl. Acad.Sci. USA 90:913-917 and in U.S. Pat. No. 5,693,507.

[0085] A plant cell, tissue, organ, or plant into which the recombinantDNA constructs containing the expression constructs have been introducedis considered transformed, transfected, or transgenic. A transgenic ortransformed cell or plant also includes progeny of the cell or plant andprogeny produced from a breeding program employing such a transgenicplant as a parent in a cross and exhibiting an altered phenotyperesulting from the presence of a KAS nucleic acid sequence.

[0086] The term “introducing,” in the context of inserting a nucleicacid sequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

[0087] Plant expression or transcription constructs having aβ-ketoacyl-ACP synthase as the DNA sequence of interest for increased ordecreased expression thereof may be employed with a wide variety ofplant life, particularly, plant life involved in the production of oilsfor edible and industrial uses. Importantly, the plant expression and/ortranscription constructs of the present invention find use in bothmonocotyledenous and dicotyledenous species, and will be readilyapplicable to new and/or improved transformation and regulationtechniques. Preferred plants include Acacia, alfalfa, aneth, apple,apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans,beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage,canola, cantaloupe, carrot, cassava, cauliflower, celery, cherry,chicory, cilantro, citrus, clementines, coffee, corn, cotton, cucumber,Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs,garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce,leeks, lemon, lime, Loblolly pine, mango, melon, mushroom, nectarine,nut, oat, oil palm, oil seed rape, okra, onion, orange, an ornamentalplant, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon,pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin,quince, radiata pine, radicchio, radish, raspberry, rice, rye,safflower, sorghum, Southern pine, soybean, spinach, squash, strawberry,sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea,tobacco, tomato, triticale, turf, turnip, a vine, watermelon, wheat,yams, and zucchini.

[0088] As used herein, the term “plant” includes reference to wholeplants, plant organs (for example, leaves, stems, roots, etc.), seeds,and plant cells and progeny of same. Plant cell, as used hereinincludes, without limitation, seeds suspension cultures, embryos,meristematic regions, callus tissue, leaves roots shoots, gametophytes,sporophytes, pollen, and microspores. The class of plants which can beused in the methods of the present invention is generally as broad asthe class of higher plants amenable to transformation techniques,including both monocotyledenous and dicotyledenous plants. Particularlypreferred plants include Brassica, soybean, and corn.

[0089] As used herein, “transgenic plant” includes reference to a plantwhich comprises within its genome a heterologous polynucleotide.Generally, the heterologous polynucleotide is stably integrated withinthe genome such that the polynucleotide is passed on to successivegenerations. The heterologous polynucleotide may be integrated into thegenome alone or as part of a recombinant expression cassette.“Transgenic” is used herein to include any cell, cell line, callus,tissue, plant part or plant, the genotype of which has been altered bythe presence of heterologous nucleic acid including those transgenicsinitially so altered as well as those created by sexual crosses orasexual propagation from the initial transgenic. The term “transgenic”as used herein does not encompass the alteration of the genome(chromosomal or extra-chromosomal) by conventional plant breedingmethods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

[0090] Thus a plant having within its cells a heterologouspolynucleotide is referred to herein as a transgenic plant. Theheterologous polynucleotide can be either stably integrated into thegenome, or can be extra-chromosomal. Preferably, the polynucleotide ofthe present invention is stably integrated into the genome such that thepolynucleotide is passed on to successive generations. Thepolynucleotide is integrated into the genome alone or as part of arecombinant expression cassette. “Transgenic” is used herein to includeany cell, cell line, callus, tissue, plant part or plant, the genotypeof which has been altered by the presence of heterologous nucleic acidsincluding those transgenics initially so altered as well as thosecreated by sexual crosses or asexual reproduction of the initialtransgenics.

[0091] As used herein, “heterologous” in reference to a nucleic acid isa nucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived, or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species, or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

[0092] As used herein, a “recombinant expression cassette” is a nucleicacid construct, generated recombinantly or synthetically, with a seriesof specified nucleic acid elements which permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid sequence to betranscribed and a promoter.

[0093] Of interest in the present invention, is the use of expressionconstructs in plants to produce a reduced level of saturated fatty acidsin the plant seed oil.

[0094] It is contemplated that the gene sequences may be synthesized,either completely or in part, especially where it is desirable toprovide plant-preferred sequences. Thus, all or a portion of the desiredstructural gene (that portion of the gene which encodes the KAS protein)may be synthesized using codons preferred by a selected host.Host-preferred codons may be determined, for example, from the codonsused most frequently in the proteins expressed in a desired hostspecies.

[0095] One skilled in the art will readily recognize that antibodypreparations, nucleic acid probes (DNA and RNA) and the like may beprepared and used to screen and recover “homologous” or “related” KASfrom a variety of plant sources. Homologous sequences are found whenthere is an identity of sequence, which may be determined uponcomparison of sequence information, nucleic acid or amino acid, orthrough hybridization reactions between a known KAS and a candidatesource. Conservative changes, such as Glu/Asp, Val/Ile, Ser/Thr, Arg/Lysand Gln/Asn may also be considered in determining sequence homology.Amino acid sequences are considered homologous by as little as 25%sequence identity between the two complete mature proteins. (Seegenerally, Doolittle, R. F., OF URFS and ORFS (University Science Books,CA., 1986.)

[0096] Thus, other β-ketoacyl-ACP synthase can be obtained from thespecific exemplified sequences provided herein. Furthermore, it will beapparent that one can obtain natural and synthetic KAS, includingmodified amino acid sequences and starting materials forsynthetic-protein modeling from the exemplified KAS and from KAS whichare obtained through the use of such exemplified sequences. Modifiedamino acid sequences include sequences which have been mutated,truncated, increased and the like, whether such sequences were partiallyor wholly synthesized. Sequences which are actually purified from plantpreparations or are identical or encode identical proteins thereto,regardless of the method used to obtain the protein or sequence, areequally considered naturally derived.

[0097] For immunological screening, antibodies to the KAS protein can beprepared by injecting rabbits or mice with the purified protein orportion thereof, such methods of preparing antibodies being well knownto those in the art. Either monoclonal or polyclonal antibodies can beproduced, although typically polyclonal antibodies are more useful forgene isolation. Western analysis may be conducted to determine that arelated protein is present in a crude extract of the desired plantspecies, as determined by cross-reaction with the antibodies to the KASprotein. When cross-reactivity is observed, genes encoding the relatedproteins are isolated by screening expression libraries representing thedesired plant species. Expression libraries can be constructed in avariety of commercially available vectors, including lambda gt11, asdescribed in Sambrook, et al. (Molecular Cloning: A Laboratory Manual,Second Edition (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.).

[0098] The nucleic acid sequences associated with the KAS proteins ofthe present invention will find many uses. For example, recombinantconstructs can be prepared which can be used as probes, or which willprovide for expression of the KAS protein in host cells to produce aready source of the enzyme and/or to modify the composition of fattyacids found therein. Other useful applications may be found when thehost cell is a plant host cell, either in vitro or in vivo.

[0099] The modification of fatty acid compositions may also affect thefluidity of plant membranes. Different lipid concentrations have beenobserved in cold-hardened plants, for example. By this invention, onemay be capable of introducing traits which will lend to chill tolerance.Constitutive or temperature inducible transcription initiationregulatory control regions may have special applications for such uses.

[0100] As discussed above, the nucleic acid sequence encoding a KAS ofthis invention may include genomic, cDNA or mRNA sequence. By “encoding”is meant that the sequence corresponds to a particular amino acidsequence either in a sense or anti-sense orientation. By“extrachromosomal” is meant that the sequence is outside of the plantgenome of which it is naturally associated. By “recombinant” is meantthat the sequence contains a genetically engineered modification throughmanipulation via mutagenesis, restriction enzymes, and the like.

[0101] Once the desired KAS nucleic acid sequence is obtained, it may bemanipulated in a variety of ways. Where the sequence involves non-codingflanking regions, the flanking regions may be subjected to resection,mutagenesis, etc. Thus, transitions, transversions, deletions, andinsertions may be performed on the naturally occurring sequence. Inaddition, all or part of the sequence may be synthesized. In thestructural gene, one or more codons may be modified to provide for amodified amino acid sequence, or one or more codon mutations may beintroduced to provide for a convenient restriction site or other purposeinvolved with construction or expression. The structural gene may befurther modified by employing synthetic adapters, linkers to introduceone or more convenient restriction sites, or the like.

[0102] The nucleic acid or amino acid sequences encoding a KAS of thisinvention may be combined with other non-native, or “heterologous”,sequences in a variety of ways. By “heterologous” sequences is meant anysequence which is not naturally found joined to the KAS, including, forexample, combinations of nucleic acid sequences from the same plantwhich are not naturally found joined together.

[0103] The DNA sequence encoding a KAS of this invention may be employedin conjunction with all or part of the gene sequences normallyassociated with the KAS. In its component parts, a DNA sequence encodingKAS is combined in a DNA construct having, in the 5′ to 3′ direction oftranscription, a transcription initiation control region capable ofpromoting transcription and translation in a host cell, the DNA sequenceencoding KAS and a transcription and translation termination region.

[0104] Potential host cells include both prokaryotic cells, such as E.coli and eukaryotic cells such as yeast, insect, amphibian, or mammaliancells. A host cell may be unicellular or found in a multicellulardifferentiated or undifferentiated organism depending upon the intendeduse. Preferably, host cells of the present invention include plantcells, both monocotyledenous and dicotyledenous. Cells of this inventionmay be distinguished by having a KAS foreign to the wild-type cellpresent therein, for example, by having a recombinant nucleic acidconstruct encoding a said foreign KAS therein.

[0105] The methods used for the transformation of the host plant cellare not critical to the present invention. The transformation of theplant is preferably nonreversible, i.e. by integration of the introducedexpression constructs into the host plant genome, so that the introducedconstructs are passed onto successive plant generations. The skilledartisan will recognize that a wide variety of transformation techniquesexist in the art, and new techniques are continually becoming available.Any technique that is suitable for the target host plant can be employedwithin the scope of the present invention. For example, the constructscan be introduced in a variety of forms including, but not limited to asa strand of DNA, in a plasmid, or in an artificial chromosome. Theintroduction of the constructs into the target plant cells can beaccomplished by a variety of techniques, including, but not limited tocalcium-phosphate-DNA co-precipitation, electroporation, microinjection,Agrobacterium infection, liposomes or microprojectile transformation.The skilled artisan can refer to the literature for details and selectsuitable techniques for use in the methods of the present invention.

[0106] Normally, included with the DNA construct will be a structuralgene having the necessary regulatory regions for expression in a hostand providing for selection of transformant cells. The gene may providefor resistance to a cytotoxic agent, e.g. antibiotic, heavy metal,toxin, etc., complementation providing prototrophy to an auxotrophichost, viral immunity or the like. Depending upon the number of differenthost species the expression construct or components thereof areintroduced, one or more markers may be employed, where differentconditions for selection are used for the different hosts.

[0107] Where Agrobacterium is used for plant cell transformation, avector may be used which may be introduced into the Agrobacterium hostfor homologous recombination with T-DNA or the Ti- or Ri-plasmid presentin the Agrobacterium host. The Ti- or Ri-plasmid containing the T-DNAfor recombination may be armed (capable of causing gall formation) ordisarmed (incapable of causing gall formation), the latter beingpermissible, so long as the vir genes are present in the transformedAgrobacterium host. The armed plasmid can give a mixture of normal plantcells and gall.

[0108] In some instances where Agrobacterium is used as the vehicle fortransforming host plant cells, the expression or transcription constructbordered by the T-DNA border region(s) will be inserted into a broadhost range vector capable of replication in E. coli and Agrobacterium,there being broad host range vectors described in the literature.Commonly used is pRK2 or derivatives thereof. See, for example, Ditta,et al., (Proc. Nat. Acad. Sci., U.S.A. (1980) 77:7347-7351) and EPA 0120 515, which are incorporated herein by reference. Alternatively, onemay insert the sequences to be expressed in plant cells into a vectorcontaining separate replication sequences, one of which stabilizes thevector in E. coli, and the other in Agrobacterium. See, for example,McBride and Summerfelt (Plant Mol. Biol. (1990) 14:269-276), wherein thepRiHRI (Jouanin, et al., Mol. Gen. Genet. (1985) 201:370-374) origin ofreplication is utilized and provides for added stability of the plantexpression vectors in host Agrobacterium cells.

[0109] Included with the expression construct and the T-DNA will be oneor more markers, which allow for selection of transformed Agrobacteriumand transformed plant cells. A number of markers have been developed foruse with plant cells, such as resistance to chloramphenicol, kanamycin,the aminoglycoside G418, hygromycin, or the like. The particular markeremployed is not essential to this invention, one or another marker beingpreferred depending on the particular host and the manner ofconstruction.

[0110] For transformation of plant cells using Agrobacterium, explantsmay be combined and incubated with the transformed Agrobacterium forsufficient time for transformation, the bacteria killed, and the plantcells cultured in an appropriate selective medium. Once callus forms,shoot formation can be encouraged by employing the appropriate planthormones in accordance with known methods and the shoots transferred torooting medium for regeneration of plants. The plants may then be grownto seed and the seed used to establish repetitive generations and forisolation of vegetable oils.

[0111] There are several possible ways to obtain the plant cells of thisinvention which contain multiple expression constructs. Any means forproducing a plant comprising a construct having a nucleic acid sequenceof the present invention, and at least one other construct havinganother DNA sequence encoding an enzyme are encompassed by the presentinvention. For example, the expression construct can be used totransform a plant at the same time as the second construct either byinclusion of both expression constructs in a single transformationvector or by using separate vectors, each of which express desiredgenes. The second construct can be introduced into a plant which hasalready been transformed with the first expression construct, oralternatively, transformed plants, one having the first construct andone having the second construct, can be crossed to bring the constructstogether in the same plant.

[0112] The present invention also provides methods for the production ofa plant having a seed oil comprising less than about 5%, preferably lessthan about 4%, more preferably less than about 3.4% saturated fattyacids as a percentage of the total fatty acids contained in the seedoil. Thus, ranges in the levels of saturated fatty acids in a seed oilrange from about 0.5% to about 5% saturated fatty acids as a percentageof total fatty acids, preferably from about 0.5% to about 4% saturatedfatty acids.

[0113] The methods for the production of a plant having the reducedlevels of saturated fatty acids comprise the expression of one or moreintroduced nucleic acid sequences encoding β-ketoacyl-ACP synthase in ahost plant cell. For a greater reduction in the levels of saturatedfatty acids, the expression of additional nucleic acid sequencesencoding proteins involved in the synthesis of fatty acids can beemployed. Such sequences include, but are not limited to: desaturaseencoding sequences, including delta 9, delta 12 and delta 15desaturases, and thioesterase sequences.

[0114] The invention now being generally described, it will be morereadily understood by reference to the following examples which areincluded for purposes of illustration only and are not intended to limitthe present invention.

EXAMPLES Example 1

[0115] Identification and Characterization of a β-Ketoacyl-ACP Synthasefrom Synechocystis

[0116] The genome sequence of the unicellular cyanobacterium,Synechocystis sp. (strain PCC6803) (Kaneko, et a. (1996) DNA Res.3(3):109-136; Kaneko, et al. (1996) DNA Res. 3(3):185-209; and Nakamura,et al. (1998) Nucleic Acids Res. 26(1):63-67) was searched with the E.coli FabF sequence (Magnuson, et al. (1995) J Bacteriol.177(12);3593-3595) encoding a β-Ketoacyi-ACP Synthase II (KASII)protein. The search identified two sequences in the Synechocystisdatabase (Nakamura, et al. (1998) supra). These sequences were used todesign synthetic oligonucleotides for use as primers in Polymerase ChainReactions (PCR) to isolate the sequence most similar to the KASII classof proteins. The primers 5′-GGATCCGCATGCATGGCAAATTTGGAAAAGAAACGTGTTGTTGTA-3′ (Synch FabF-F, SEQ ID NO: 3) and 5′-GGATCCAAGCTTCTATTGATATTTTTTGAAAGCTAAGG-3′ (Synch FabF-R, SEQ ID NO: 4) wereused in PCR reactions with complementary DNA (CDNA) obtained fromSynechocystis sp (strain PCC6803). A single reaction product ofapproximately 1.25 kb was digested with SphI and HindIII and cloned intothe respective sites of pQE30 (Invitrogen, Carlsbad, Calif.) to createthe construct pCGN8386 for expression in E. coli. The sequence wasconfirmed by sequencing on an automated sequencer, and is provided inSEQ ID NO: 1. The deduced amino acid sequence of the Synechocystis KASis provided in SEQ ID NO: 2.

[0117] The E. coli expression vector pCGN8386 is transformed into E.coli, and recombinant protein is affinity purified and analyzed forenzyme activity using methods described by Edwards, et al. (1997) FEBSLetters 402:62-66. The results of the enzyme assay are provided in Table1 below. TABLE 1 12:0 16:0 16:1 16:0/16:1 E coli FabB 1.00 0.02 0.030.67 Syn FabF 1.00 0.15 0.30 0.48 E coil FabF 1.00 0.03 0.30 0.02

[0118] Results of the enzyme activity assay indicate that except for ahigher activity on 16:0-ACP substrates, the Synechocystis KAS has aprofile identical to that of the FabF enzyme of E. coli.

Example 2

[0119] Complementation of E coli FabF Mutants

[0120] The Synechocystis FabF sequence was analyzed for complementationof the mutant E coli FabF (Garwin, et al. (1980) J Biol Chem255(8):3263-3265). Expression of the Syn FabF did not complement theFabF mutation in E. coli.

[0121] Comparisons of the Syn FabF crystal structure and the E. coliFabF structure (Moche, et al. (2000) J. Mol. Biol.) shows that the majordifference between the two is the substitution of glycine (G) atposition 202 to serine (S) and phenylalanine (F) at position 137 tomethionine (M) in the Syn FabF.

[0122] To investigate the role of these mutations in the Syn FabF,mutations were introduced into the Syn FabF sequence that convert S202to G and M137 to F. Expression of these two mutated Syn FabF sequenceswere able to complement the E. coli mutant.

Example 3

[0123] Plant Expression Construct Preparation

[0124] A series of expression constructs are prepared for transformationinto various plants, either alone, or in combination with additionalsequences encoding proteins involved in fatty acid biosynthesis.

[0125] The construct pCGN8378 is a double napin expression cassette forthe seed preferential expression of the Cuphea pullcherrima KASI (cpuKASB/7-8, described in PCT Publication WO 98/46776, the entirety of whichis incorporated herein by reference) and KASIV (cpuKAS A/p7-6A,described in PCT Publication WO 98/46776, the entirety of which isincorporated herein by reference) sequences in Brassica.

[0126] In addition, a double expression cassette construct, pCGN9807,was prepared to express the C. Pullcherrimma KASI and KASIV sequencesfrom promoters derived from the soybean a′ subunit of b-conglycinin (soy7s, (Chen et al., (1986), Proc. Natl. Acad. Sci., 83:8560-8564)) fortransformation into soybean cells. The pCGN9807 construct provides forthe seed preferential expression of the KAS sequences in the soybeanseed cells.

[0127] The plant expression construct, pCGN3231, for the expression ofthe safflower Delta-9 desaturase from the napin promoter sequence is asdescribed in U.S. Pat. No. 5,723,595, the entirety of which isincorporated herein by reference.

[0128] Another construct, the safflower Delta-9 desaturase expressionconstruct, is also prepared for the seed preferential expression of thesequence from the 7S promoter. This construct, pCGN9373, contains thesafflower delta-9 desaturase coding sequence under the control of thesoy 7S promoter and the pea rbcS termination sequences.

[0129] For expression of the Syn FabF sequence in soybean, a constructwas prepared to express the FabF sequence in combination with a delta-9desaturase sequence from safflower (G. Thompson, et al., Primarystructure of the precursor and mature forms of stearoyl-acyl carrierprotein desaturase from safflower embryos and requirement of ferredoxinfor enzyme activity, Proc. Natl. Acad. Sci. USA, Vol 88, pp 2578-2582,March 1991). The construct was prepared by first PCR amplifying atransit peptide from Cuphea hookeriana KASII-7 (described in PCTPublication WO 98/46776) using the primers KASI1-7 tpF5′-CTGAGATCTGTCGACATGGCGACCGCTT CTCGC-3′ (SEQ ID NO: 5) and KASII-7 tpR5′-GACAGATCTTGTGGAGA CTTCCTGTGCAGG-3′ (SEQ ID NO: 6). The resultingfragment was digested with Bg/II and placed 5′ (upstream) of the SynFabF cDNA by cloning into the BamHI site of pCGN8386 to create pCGN9382.A SaII/HindIII fillin fragment from pCGN9382 containing the ChKASII-7/Syn FabF was cloned into the XhoI/EcoRI fillin pCGN3892 betweenthe 7S promoter and pea rbcS terminator to generate pCGN9868.

[0130] A second construct for the expression of the delta-9 desaturasesequence was also prepared. The safflower delta-9 desaturase wasdigested from pCGN3231 (described in U.S. Pat. No. 5,723,595, theentirety of which is incorporated herein by reference) as a XhoIfragment into the same site of pCGN3892 7S expression cassette to createpCGN9359. A NotI fragment containing the delta-9 desaturase expressioncassette from pCGN9359 was ligated into the Notl site of pMON33510 togenerate the vector pCGN9882. The plant transformation constructpCGN9883 was created by ligating a SaII/SwaI fragment containing the SynFabF transit peptide fusion from pCGN9868 into the SnsBI/SaII ofpCGN9882.

Example 4

[0131] Complementation of Arabidopsis FabF Mutants

[0132] Expression of the Syn FabF coding sequence in a Fab1 mutant lineof Arabidopsis (Wu, et al. (1997) Plant Physiol. 113(2):347-356)complemented the phenotype. Mutant lines expressing the Syn FabFdemonstrate a level of 16:0 reduced from the 16% of the mutant tosimilar to wildtype Arabidopsis (8%). Thus, the Synechocystis KASsequence is able to complement the Arabidopsis fab1 mutant phenotype.

Example 5

[0133] Transgenic Plant Analysis

[0134] 5A. Transgenic Brassica Oils Analysis

[0135] Transgenic Brassica plants containing the plant expressionconstruct pCGN3231 were crossed with transgenic Brassica plantscontaining the expression construct pCGN8378. Fatty acid compositionalanalysis was performed using gas chromatography of fatty acid methylesters (FAME) derived from seed oil obtained from transgenic Brassicalines. Results of the analysis are provided in Table 2 as well as inFIG. 1. The total saturates column is the total of the C6:0, C8:0,C10:0, C12:0, C14:0, C16:0, C18:0, C20:0, C22:0 and C24:0 fatty acidsfound in the oil. TABLE 2 Total Plant Line C16:0 C18:0 C20:0 C20:1Saturates DH37-4 1.74 1.00 0.30 3.84 3.19 DH37-5 1.62 1.04 0.30 4.223.10 DH95-1 1.76 0.83 0.34 6.27 3.17 DH95-2 1.69 0.88 0.38 6.68 3.21DH95-6 1.65 0.86 0.38 6.75 3.13 DH97-2 1.89 0.94 0.27 3.21 3.20 DH97-31.90 0.96 0.28 3.23 3.29 DH135-2 1.66 1.19 0.33 6.93 3.35 DH135-4 1.601.10 0.30 6.57 3.12 DH199-2 1.56 0.96 0.29 6.83 3.04 DH199-4 1.60 0.960.29 6.39 3.12 DH229-1 1.69 1.01 0.30 5.62 3.16 DH240-2 1.90 0.84 0.335.81 3.38 DH240-4 1.70 0.88 0.33 5.95 3.21 DH266-6 1.82 0.83 0.34 5.673.31 DH561-1 1.75 0.98 0.40 7.30 3.35 DH561-6 1.81 0.92 0.39 5.01 3.35DH591-2 1.70 0.91 0.37 5.15 3.29 DH591-6 1.71 0.90 0.39 5.16 3.34

[0136] Total level of saturated fatty acids in oil obtained from seedcontaining the Cuphea pulicherima KASI and KAS IV sequences as well asthe safflower delta-9 desaturase demonstrate a significantly decreasedamount of total saturates as compared to non-transformed controlBrassica lines. Levels of total saturated fatty acids are reduced toabout 3.0 wt %, and below 3.4 wt %, while the levels of total saturatedfatty acids obtained in non-transformed controls lines are about 6.0 wt%.

[0137] 5B. Transgenic Soybean Oils Analysis

[0138] Seed oil obtained from transgenic soybean plants containing theconstruct pCGN9807 are analyzed for fatty acid composition using gaschromatography of fatty acid methyl esters. The results show that thetransgenic 9807 lines demonstrate a reduced amount of total saturatedfatty acids compared to non-transgenic wild-type (FIG. 2). Inparticular, levels of C16:0 fatty acids are reduced to less than 2.5 wt%, compared to normal levels of about 13 wt % (FIG. 3). In addition, thelevels of C20:1 are also increased in the seed oils of transgenic plantscontaining the KASI and KASIV sequences (FIG. 4).

[0139] Transgenic soybean plants containing the expression constructpCGN9883 were analyzed for fatty acid composition using gaschromatography of fatty acid methyl esters (FAME) derived from oilsobtained from various lines. Oil analyses data from T2 segregationgeneration of soybean seed showed that the levels of 16:0 has droppedfrom between about 13 and about 16% in non-transgenic control lines toabout 2.1% in the seed oils of transgenic soybean plants. These levelsare as low as those obtained with the combined expression of KASI(B) andKASIV(A) enzyme shown above. Thus, expression of the Synechocystis fabFalone can decrease the levels of saturated fat in a host plant cell andcan be combined with delta 9 desaturase to further reduce the amount ofsaturated fatty acids in a host cell. To bring the levels of saturatedfat to ≦3.2 wt % as defined by FDA, one may need to include the strategyof silencing FatB thioestearse from soy by sense and/or anti-sensing theintrons, negative dominant mutations, and targeting the FatB promoter tointerfere with the expression of this gene.

[0140] The above results demonstrate that the Synechocystis FabFsequence can be used in the preparation of constructs for expression inhost cells. Furthermore, expression of the KAS sequences, either aloneor in combination with additional sequences encoding proteins involvedin fatty acid biosynthesis can be used in the preparation of transgenicplants having reduced saturated fatty acid content.

[0141] In light of the detailed description of the invention and theexamples presented above, it can be appreciated that the several aspectsof the invention are achieved.

[0142] It is to be understood that the present invention has beendescribed in detail by way of illustration and example in order toacquaint others skilled in the art with the invention, its principles,and its practical application. Particular formulations and processes ofthe present invention are not limited to the descriptions of thespecific embodiments presented, but rather the descriptions and examplesshould be viewed in terms of the claims that follow and theirequivalents. While some of the examples and descriptions above includesome conclusions about the way the invention may function, the inventorsdo not intend to be bound by those conclusions and functions, but putthem forth only as possible explanations.

[0143] It is to be further understood that the specific embodiments ofthe present invention as set forth are not intended as being exhaustiveor limiting of the invention, and that many alternatives, modifications,and variations will be apparent to those of ordinary skill in the art inlight of the foregoing examples and detailed description. Accordingly,this invention is intended to embrace all such alternatives,modifications, and variations that fall within the spirit and scope ofthe following claims.

1 6 1 1275 DNA Synechocystis sp. 1 ggatccgcat gcatggcaaa tttggaaaagaaacgtgttg ttgtaacggg attgggagcc 60 atcaccccca tcggtaatac tctccaagactattggcaag gcttaatgga gggtcgtaac 120 ggcattggcc ccattacccg tttcgatgctagtgaccaag cctgccgttt tggaggggaa 180 gtaaaggatt ttgatgctac ccagtttcttgaccgcaaag aagctaaacg gatggaccgg 240 ttttgccatt ttgctgtttg tgccagtcaacaggcaatta acgatgctaa gttggtgatt 300 aacgaactca atgccgatga aatcggggtattgattggca cgggcattgg tggtttgaaa 360 gtactggaag atcaacaaac cattctgttggataagggtc ctagccgttg cagtcctttt 420 atgatcccga tgatgatcgc caacatggcctctgggttaa ccgccatcaa cttaggggcc 480 aagggtccca ataactgtac ggtgacggcctgtgcggcgg gttccaatgc cattggagat 540 gcgtttcgtt tggtgcaaaa tggctatgctaaggcaatga tttgcggtgg cacggaagcg 600 gccattaccc cgctgagcta tgcaggttttgcttcggccc gggctttatc tttccgcaat 660 gatgatcccc tccatgccag tcgtcccttcgataaggacc gggatggttt tgtgatgggg 720 gaaggatcgg gcattttgat cctagaagaattggaatccg ccttggcccg gggagcaaaa 780 atttatgggg aaatggtggg ctatgccatgacctgtgatg cctatcacat taccgcccca 840 gtgccggatg gtcggggagc caccagggcgatcgcctggg ccttaaaaga cagcggattg 900 aaaccggaaa tggtcagtta catcaatgcccatggtacca gcacccctgc taacgatgtg 960 acggaaaccc gtgccattaa acaggcgttgggaaatcatg cctacaatat tgcggttagt 1020 tctactaagt ctatgaccgg tcacttgttgggcggctccg gaggtatcga agcggtggcc 1080 accgtaatgg cgatcgccga agataaggtaccccccacca ttaatttgga gaaccccgac 1140 cctgagtgtg atttggatta tgtgccggggcagagtcggg ctttaatagt ggatgtagcc 1200 ctatccaact cctttggttt tggtggccataacgtcacct tagctttcaa aaaatatcaa 1260 tagaagcttg gatcc 1275 2 416 PRTSynechocystis sp. 2 Met Ala Asn Leu Glu Lys Lys Arg Val Val Val Thr GlyLeu Gly Ala 1 5 10 15 Ile Thr Pro Ile Gly Asn Thr Leu Gln Asp Tyr TrpGln Gly Leu Met 20 25 30 Glu Gly Arg Asn Gly Ile Gly Pro Ile Thr Arg PheAsp Ala Ser Asp 35 40 45 Gln Ala Cys Arg Phe Gly Gly Glu Val Lys Asp PheAsp Ala Thr Gln 50 55 60 Phe Leu Asp Arg Lys Glu Ala Lys Arg Met Asp ArgPhe Cys His Phe 65 70 75 80 Ala Val Cys Ala Ser Gln Gln Ala Ile Asn AspAla Lys Leu Val Ile 85 90 95 Asn Glu Leu Asn Ala Asp Glu Ile Gly Val LeuIle Gly Thr Gly Ile 100 105 110 Gly Gly Leu Lys Val Leu Glu Asp Gln GlnThr Ile Leu Leu Asp Lys 115 120 125 Gly Pro Ser Arg Cys Ser Pro Phe MetIle Pro Met Met Ile Ala Asn 130 135 140 Met Ala Ser Gly Leu Thr Ala IleAsn Leu Gly Ala Lys Gly Pro Asn 145 150 155 160 Asn Cys Thr Val Thr AlaCys Ala Ala Gly Ser Asn Ala Ile Gly Asp 165 170 175 Ala Phe Arg Leu ValGln Asn Gly Tyr Ala Lys Ala Met Ile Cys Gly 180 185 190 Gly Thr Glu AlaAla Ile Thr Pro Leu Ser Tyr Ala Gly Phe Ala Ser 195 200 205 Ala Arg AlaLeu Ser Phe Arg Asn Asp Asp Pro Leu His Ala Ser Arg 210 215 220 Pro PheAsp Lys Asp Arg Asp Gly Phe Val Met Gly Glu Gly Ser Gly 225 230 235 240Ile Leu Ile Leu Glu Glu Leu Glu Ser Ala Leu Ala Arg Gly Ala Lys 245 250255 Ile Tyr Gly Glu Met Val Gly Tyr Ala Met Thr Cys Asp Ala Tyr His 260265 270 Ile Thr Ala Pro Val Pro Asp Gly Arg Gly Ala Thr Arg Ala Ile Ala275 280 285 Trp Ala Leu Lys Asp Ser Gly Leu Lys Pro Glu Met Val Ser TyrIle 290 295 300 Asn Ala His Gly Thr Ser Thr Pro Ala Asn Asp Val Thr GluThr Arg 305 310 315 320 Ala Ile Lys Gln Ala Leu Gly Asn His Ala Tyr AsnIle Ala Val Ser 325 330 335 Ser Thr Lys Ser Met Thr Gly His Leu Leu GlyGly Ser Gly Gly Ile 340 345 350 Glu Ala Val Ala Thr Val Met Ala Ile AlaGlu Asp Lys Val Pro Pro 355 360 365 Thr Ile Asn Leu Glu Asn Pro Asp ProGlu Cys Asp Leu Asp Tyr Val 370 375 380 Pro Gly Gln Ser Arg Ala Leu IleVal Asp Val Ala Leu Ser Asn Ser 385 390 395 400 Phe Gly Phe Gly Gly HisAsn Val Thr Leu Ala Phe Lys Lys Tyr Gln 405 410 415 3 45 DNASynechocystis sp. 3 ggatccgcat gcatggcaaa tttggaaaag aaacgtgttg ttgta 454 38 DNA Synechocystis sp. 4 ggatccaagc ttctattgat attttttgaa agctaagg38 5 33 DNA Cuphea hookeriana 5 ctgagatctg tcgacatggc gaccgcttct cgc 336 30 DNA Cuphea hookeriana 6 gacagatctt gtggagactt cctgtgcagg 30

What is claimed is:
 1. An isolated polynucleotide selected from thegroup consisting of: a) a nucleotide sequence encoding the polypeptideof SEQ ID NO: 2; b) a nucleotide sequence comprising SEQ ID NO: 1; c) anucleotide sequence which has at least about 70% identity to that of SEQID NO: 1 over the entire length of SEQ ID NO: 1; d) a nucleotidesequence that hybidrizes, under stringent conditions, to SEQ. ID NO: 1or a fragment thereof; and e) a nucleotide sequence complementary to thenucleotide sequence of (a), (b), (c), or (d); wherein the polynucleotideencodes a polypeptide having KAS activity.
 2. An isolated polynucleotideof claim 1 comprising SEQ ID NO:
 1. 3. An isolated polynucleotide ofclaim 1 comprising a nucleotide sequence which has at least about 70%identity to that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.4. An isolated polynucleotide of claim 1 comprising a nucleotidesequence which has at least about 80% identity to that of SEQ ID NO: 1over the entire length of SEQ ID NO:
 1. 5. An isolated polynucleotide ofclaim 1 comprising a nucleotide sequence which has at least about 90%identity to that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.6. An isolated polynucleotide of claim 1 comprising a nucleotidesequence which has at least about 95% identity to that of SEQ ID NO: 1over the entire length of SEQ ID NO:
 1. 7. An isolated polynucleotide ofclaim 1 that hybridizes, under stringent conditions, to SEQ ID NO: 1 ora fragment thereof.
 8. As isolated polynucleotide according to claim 7that hybridizes to SEQ ID NO: 1 under the following set of stringentconditions: a) overnight incubation at 42° C. in a solution comprising;b) 50% formamide, 5×SSC; c) 50 mM sodium phosphate; d) 5×Denhardt'ssolution; e) 10% dextran sulfate; f) 20 micrograms/milliliter denatured,sheared salmon sperm DNA; g) followed by washing the hybridizationsupport in 0.1×SSC at approximately 65° C.
 9. A polynucleotide; whereinsaid polynucleotide comprises the formula: X—(R₁)_(n)—(R₂)—(R₃)_(n)—Ywherein, at the 5′ end, X is hydrogen; and at the 3′ end, Y is hydrogenor a metal; R₁ and R₃ are any nucleic acid residue; n is an integerbetween 1 and about 3000; and R₂ is the nucleic acid sequence set forthin SEQ ID NO:
 1. 10. A nucleic acid construct comprising a promoterfunctional in a host cell operably linked to the polynucleotide ofclaim
 1. 11. A nucleic acid construct according to claim 10, whereinsaid polynucleotide is operably linked in an orientation relative tosaid promoter selected from the group consisting of sense and antisense.12. A nucleic acid construct according to claim 11, wherein saidpolynucleotide is operably linked to a construct encoding for adesaturase enzyme.
 13. The nucleic acid construct according to claim 12,wherein said construct encoding for a desaturase enzyme encodes for adelta-9 desaturase enzyme.
 14. A host cell modified by introducing thenucleic acid construct of claim
 10. 15. The host cell of claim 14,wherein said host cell is a plant host cell.
 16. A transgenic plant, orany part thereof, comprising the host cell of claim
 15. 17. Thetransgenic plant, or any part thereof, of claim 16, wherein said plantis selected from the group consisting of Brassica, soybean and corn. 18.A seed from the transgenic plant of claim
 16. 19. A progeny from thetransgenic plant of claim
 16. 20. A seed from the progeny of claim 19.21. A plant, or any part thereof, from the seed of claim
 18. 22. Amethod for modifying the saturated fatty acid content in a recombinanthost cell, comprising: a) transforming or transfecting a cell; b)wherein said cell becomes the recombinant host cell; and wherein c) saidtransformation or transfection occurs with a nucleic acid constructcomprising a transcriptional initiation region and a polynucleotidesequence encoding β-ketoacyl-ACP synthase; d) such that said host cellproduces a β-ketoacyl-ACP synthase and thereby modifies the saturatedfatty acid content in said host cell.
 23. A method for increasing theexpression of β-ketoacyl-ACP synthase in a recombinant host cell,comprising: a) transforming or transfecting a cell; b) wherein said cellbecomes the recombinant host cell; and wherein c) said transformation ortransfection occurs with a nucleic acid construct comprising atranscriptional initiation region and a polynucleotide sequence encodingβ-ketoacyl-ACP synthase; d) such that said host cell produces aβ-ketoacyl-ACP synthase and thereby increases expression ofβ-ketoacyl-ACP synthase.
 24. A method for increasing the copy number ofnucleic acid constructs which encode β-ketoacyl-ACP synthase in arecombinant host cell, comprising: a) transforming or transfecting acell; b) wherein said cell becomes the recombinant host cell; andwherein c) said transformation or transfection occurs with a nucleicacid construct comprising a transcriptional initiation region and apolynucleotide sequence encoding β-ketoacyl-ACP synthase.
 25. The methodof claim 22, 23 or 24 wherein said β-ketoacyl-ACP synthase comprises anamino acid having at least about 70% identity to SEQ ID NO:
 2. 26. Themethod according to claim 22 wherein said host cell is selected from thegroup consisting of plant cells, bacterial cells, yeast cells, and algalcells.
 27. The method according to claim 22 wherein said modification ofsaturated fatty acids is a reduction in total saturated fatty acids. 28.The method according to claim 22, wherein said modification of saturatedfatty acids is a reduction in C16:0 fatty acids.
 29. The methodaccording to claim 22, wherein said modification of saturated fattyacids is a reduction of total fatty acids to a level less than about 3.5weight percent.
 30. An oil produced by the method according to claim 29.31. A plant according to claim 17; wherein said plant consist of asoybean seed.
 32. A soybean seed according to claim 35; wherein saidseed contains less than about 3.5% weight percent saturated fatty acid.33. A plant according to claim 17; wherein said plant consists of a cornseed.
 34. A corn seed according to claim 33; wherein said seed containsless than about 3.5% weight percent saturated fatty acid.
 35. A plantaccording to claim 17; wherein said plant consists of a Brassica seed.36. A Brassica seed according to claim 35; wherein said seed containsless than about 3.5% weight percent saturated fatty acid.